Dietary factors have long been understood to play a critical role in the development of diseases, such as atherosclerosis and insulin resistance. As recent evidence indicates that chronic inflammatory processes also underpin the development of these diseases, current research focuses on the potential mechanisms that link nutrition and inflammatory signalling. It has been shown that the ingestion of fatty meals is associated with the transient activation of circulating monocytes and increases in circulating inflammatory markers, such as TNF-α and IL-6. These responses have been found to be due to the induction of mild post-prandial endotoxaemia following a fatty meal in human subjects and in animal models. Not only endotoxin (also referred to as lipopolysaccharide, LPS), but also bacterial lipopeptides (BLP) and other circulating pathogen-associated molecular patterns (PAMPs) may promote inflammation observed in the post-prandial phase.
PAMPs are recognised by pattern recognition receptors in plants and mammals. Some PAMPs are recognised by Toll-like receptors (TLRs). TLRs are a family of receptors known to elicit innate immune activation of mammalian monocytes. Each member of the TLR family recognises its specific repertoire of ligands. For example, TLR-4 recognises LPS, whereas TLR-2 recognises BLP, and so on. Some PAMPS are recognised by nucleotide-binding oligomerisation domain (NOD)-like receptors (NLR), such as NOD1 and NOD2.
To date, it has been widely considered that the source of the circulating endotoxins thought to cause postprandial inflammation (i.e. after eating a meal) is the resident intestinal microflora in the mammal. It has been recently discovered that the likely site of diet-induced LPS translocation is at the small intestine. However, the small intestine contains only very low levels of endogenous bacteria. Furthermore, recent findings suggest that chylomicrons are the likely vehicle for endotoxin translocation in response to a fatty meal.
The inventor has reconsidered the conventional understanding of the sources of endotoxins which are thought to cause inflammation, and investigated whether common foodstuffs may contain appreciable quantities of endotoxins, or other agents, such as Toll-like receptor (TLR) stimulants or nucleotide-binding oligomerisation domain (NOD)-like receptor (NLR) stimulants, that may be capable of eliciting innate immune activation of human monocytes. However, measurement of the concentrations of TLR-stimulants or NLR-stimulants in food products presents several difficulties. For example, as TLR-stimulants or NLR stimulants can be derived from any type of micro-organism, they show an inherently large antigenic and molecular diversity, which precludes the use of traditional ELISA or mass-spectrometry techniques. The most widely used assay for the detection of endotoxins in foodstuffs is the limulus-amoebocyte-lysate (LAL) assay. However, the inventor has found that the LAL assay is not suitable for measuring the quantities of endotoxins, Toll-like receptors (TLRs) stimulants or NOD-like receptor (NLR) stimulants for several reasons.
Firstly, it is well-established that the limulus assay generates a positive reaction to β-glucans which can be common in foodstuffs, thereby potentially generating false-positive results. Secondly, the inventor has found that several forms of non-enterobacterial lipid-A, which can often be antagonists of TLR-4 and LPS-signalling in human cells, stimulate a positive reaction in the limulus assay. Notably, many environmental and food-borne organisms possess a non-enterobacterial lipid-A structure which does not stimulate human TLR-4/MD2. Thirdly, as the LAL assay is insensitive to lipopeptides and flagellins, it cannot be used to quantify these PAMPs.
It will therefore be appreciated that there are a number of problems associated with currently available assays for detecting the inflammatory risk posed by foodstuffs. Accordingly, there is a need for improved assays, which accurately, rapidly and conveniently assess the inflammatory risk of foodstuffs. In addition, to determining the inflammatory risk of foodstuffs, there is also a need for improved assays which assess the inflammatory risk caused by saliva, and other orally-ingestible samples. Accordingly, the inventor set out to develop an assay for determining the risk of orally-ingestible biological samples, such as saliva or foodstuffs, for causing inflammation.